PROJECT SUMMARY Histone PTMs are implicated in a variety of nuclear processes including gene regulation, DNA repair and chromatin structure. Misregulation of these chemical decorations have immense consequences for human biology and have the potential to drive disease states. Recently, we have uncovered a new histone PTM that involves the covalent addition of the neurotransmitter, serotonin, on to the N-terminal tail of histone H3. Interestingly, in compelling preliminary data, we have shown that the installation of serotonin onto glutamine 5 is catalyzed by the enzyme tissue transglutaminase 2 (TGM2). Importantly, the misregulation of TGM2 has been implicated in cancer and neurodegenerative disease highlighting its importance. Our preliminary data also highlights the use of a newly developed, highly specific validated antibody for H3Q5ser, to show that serotonylation is highly enriched in serotonin-producing organisms and that the modification is increased in post-differentiated raphe-medulloblastoma cells (RN46A-B14). While the mechanism by which serotonin is added has been uncovered, its role while present on H3 is unknown. The major goal of this proposal is to shed light on the function of H3Q5ser and also interrogate if it is a dynamic PTM. To do this, I plan to employ semisynthetic techniques that have been developed and optimized within the Muir lab to chemically construct serotonylated mononucleosomes containing a photo crosslinker. Once in hand, I will utilize them to capture the proteins that closely interact with the H3Q5ser mark, termed `readers'. This experiment will serve as a major benchmark to begin to understand how H3Q5ser contributes to regulate the functional output of chromatin. The second major goal of this research program is to determine if H3Q5ser can be removed, and if so, how? Interestingly, TGM2, the same enzyme responsible for serotonin deposition, is known to possess deaminase activity. If H3Q5ser is removed by TGM2 then a point mutation (Gln to Glu) would be left in its place. This possibility is particularly interesting giving the propensity of histone tail mutations in cancer. Together, these experiments aim to elucidate the role of H3Q5ser as a new histone H3 PTM. The data generated by the proposed research will be of immense interest to the chromatin and chemical biology community in illuminating the function of a novel histone PTM.